Phenylbenzimidazole derivatives as ligands for GABA receptors

ABSTRACT

The present patent application discloses compounds having formula (I) ##STR1## or a pharmaceutically acceptable salt thereof or an oxide thereof, wherein R 3  is (II), ##STR2## wherein A, B and D each is CH, or one or two of A, B and D is N and the others are CH; R 11  is phenyl, benzimidazolyl, or monocyclic heteroaryl all of which may be substituted one or more times with substituents selected from alkyl, alkoxy, phenyl, halogen, CF 3 , amino, nitro, cyano, acyl, acylamino, phenyl and monocyclic heteroaryl; and one of R 6  and R 7  is hydrogen and the other is furanyl or isoxazolyl each of which may be substituted one or more times with substituents selected from halogen, alkyl, alkoxy and phenyl. The compounds are useful for the treatment of various central nervous system disorders such as epilepsy and other convulsive disorders, anxiety, sleep disorders and memory disorders.

This is an application under 35 U.S.C. §371 of PCT/GB98/00322 filed onFeb. 2, 1998.

The present invention relates to a class of substituted benzimidazolederivatives and to their use in therapy. More particularly, thisinvention is concerned with substituted 1-phenylbenzimidazolederivatives which are ligands for GABA_(A) receptors and are thereforeuseful in the therapy of deleterious mental states.

Receptors for the major inhibitory neurotransmitter, gamma-aminobutyricacid (GABA), are divided into two main classes: (1) GABA_(A) receptors,which are members of the ligand-gated ion channel superfamily; and (2)GABA_(B) receptors, which may be members of the G-protein linkedreceptor superfamily. Since the first cDNAs encoding individual GABA_(A)receptor subunits were cloned the number of known members of themammalian family has grown to include at least six α subunits, four βsubunits, three γ subunits, one δ subunit, one ε subunit and two ρsubunits.

Although knowledge of the diversity of the GABA_(A) receptor gene familyrepresents a huge step forward in our understanding of this ligand-gatedion channel, insight into the extent of subtype diversity is still at anearly stage. It has been indicated that an α subunit, a β subunit and aγ subunit constitute the minimum requirement for forming a fullyfunctional GABA_(A) receptor expressed by transiently transfecting cDNAsinto cells. As indicated above, δ, ε and ρ subunits also exist, but arepresent only to a minor extent in GABA_(A) receptor populations.

Studies of receptor size and visualisation by electron microscopyconclude that, like other members of the ligand-gated ion channelfamily, the native GABA_(A) receptor exists in pentameric form. Theselection of at least one α, one β and one γ subunit from a repertoireof seventeen allows for the possible existence of more than 10,000pentameric subunit combinations. Moreover, this calculation overlooksthe additional permutations that would be possible if the arrangement ofsubunits around the ion channel had no constraints (i.e. there could be120 possible variants for a receptor composed of five differentsubunits).

Receptor subtype assemblies which do exist include, amongst many others,α1β2γ2, α2β2/3γ2, α3βγ2/3, α2βγ1, α4βδ, α5β3γ2/3, α6βγ2 and α6βδ.Subtype assemblies containing an α1 subunit are present in most areas ofthe brain and are thought to account for over 40% of GABA_(A) receptorsin the rat. Subtype assemblies containing α2 and α3 subunitsrespectively are thought to account for about 25% and 17% of GABA_(A)receptors in the rat. Subtype assemblies containing an α5 subunit areexpressed predominantly in the hippocampus and cortex and are thought torepresent about 4% of GABA_(A) receptors in the rat.

A characteristic property of all known GABA_(A) receptors is thepresence of a number of modulatory sites, one of which is thebenzodiazepine (BZ) binding site. The BZ binding site is the mostexplored of the GABA_(A) receptor modulatory sites, and is the sitethrough which anxiolytic drugs such as diazepam and temazepam exerttheir effect. Before the cloning of the GABA_(A) receptor gene family,the benzodiazepine binding site was historically subdivided into twosubtypes, BZ1 and BZ2, on the basis of radioligand binding studies. TheBZ1 subtype has been shown to be pharmacologically equivalent to aGABA_(A) receptor comprising the α1 subunit in combination with a βsubunit and γ2. This is the most abundant GABA_(A) receptor subtype, andis believed to represent almost half of all GABA_(A) receptors in thebrain.

Two other major populations are the α2βγ2 and α3βγ2/3 subtypes. Togetherthese constitute approximately a further 35% of the total GABA_(A)receptor repertoire. Pharmacologically this combination appears to beequivalent to the BZ2 subtype as defined previously by radioligandbinding, although the BZ2 subtype may also include certain α5-containingsubtype assemblies. The physiological role of these subtypes hashitherto been unclear because no sufficiently selective agonists orantagonists were known.

It is now believed that agents acting as BZ agonists at α1βγ2, α2βγ2 orα3βγ2 subunits will possess desirable anxiolytic properties. Compoundswhich are modulators of the benzodiazepine binding site of the GABA_(A)receptor by acting as BZ agonists are referred to hereinafter as"GABA_(A) receptor agonists". The α1-selective GABA_(A) receptoragonists alpidem and zolpidem are clinically prescribed as hypnoticagents, suggesting that at least some of the sedation associated withknown anxiolytic drugs which act at the BZ1 binding site is mediatedthrough GABA_(A) receptors containing the α1 subunit. Accordingly, it isconsidered that GABA_(A) receptor agonists which interact morefavourably with the α2 and/or α3 subunit than with α1 will be effectivein the treatment of anxiety with a reduced propensity to cause sedation.Also, agents which are antagonists or inverse agonists at α1 might beemployed to reverse sedation or hypnosis caused by α1 agonists.

The compounds of the present invention, being selective ligands forGABA_(A) receptors, are therefore of use in the treatment and/orprevention of a variety of disorders of the central nervous system. Suchdisorders include anxiety disorders, such as panic disorder with orwithout agoraphobia, agoraphobia without history of panic disorder,animal and other phobias including social phobias, obsessive-compulsivedisorder, stress disorders including post-traumatic and acute stressdisorder, and generalized or substance-induced anxiety disorder;neuroses; convulsions; migraine; depressive or bipolar disorders, forexample single-episode or recurrent major depressive disorder, dysthymicdisorder, bipolar I and bipolar II manic disorders, and cyclothymicdisorder; psychotic disorders including schizophrenia; neurodegenerationarising from cerebral ischemia; attention deficit hyperactivitydisorder; and disorders of circadian rhythm, e.g. in subjects sufferingfrom the effects of jet lag or shift work.

Further disorders for which selective ligands for GABA_(A) receptors maybe of benefit include pain and nociception; emesis, including acute,delayed and anticipatory emesis, in particular emesis induced bychemotherapy or radiation, as well as post-operative nausea andvomiting, and muscle spasm or spasticity, e.g. in paraplegic patients.Selective ligands for GABA_(A) receptors may also be effective aspre-medication prior to anaesthesia or minor procedures such asendoscopy, including gastric endoscopy.

EP-A-0616807 describes a class of benzimidazole derivatives substitutedat the 1-position by inter alia a phenyl moiety which in turn issubstituted at the meta position by an optionally substituted phenyl,benzimidazolyl or 5- or 6-membered monocyclic heteroaromatic group, orby an alkoxy or acyl group. These compounds are stated to possess potentbenzodiazepine receptor affinity, and thus to be useful in the treatmentof convulsions, anxiety, sleep disorders, memory disorders and otherdisorders sensitive to benzodiazepine receptor binding activity. Thereis, however, no disclosure nor any suggestion in EP-A-0616807 that theprecisely defined range of substituents prescribed for the meta positionof the phenyl moiety might be replaced by any other substituent.

The present invention provides a class of benzimidazole derivativeswhich possess desirable binding properties at various GABA_(A) receptorsubtypes. The compounds in accordance with the present invention havegood affinity as ligands for the α2 and/or α3 subunit of the humanGABA_(A) receptor. The compounds of this invention may interact morefavourably with the α2 and/or α3 subunit than with the α1 subunit.Desirably, the compounds of the invention will exhibit functionalselectivity in terms of a selective efficacy for the α2 and/or α3subunit relative to the α1 subunit.

The compounds of the present invention are GABA_(A) receptor subtypeligands having a binding affinity (K_(i)) for the α2 and/or α3 subunit,as measured in the assay described hereinbelow, of 100 nM or less,typically of 50 nM or less, and ideally of 10 nM or less. The compoundsin accordance with this invention may possess at least a 2-fold,suitably at least a 5-fold, and advantageously at least a 10-fold,selective affinity for the α2 and/or α3 subunit relative to the α1subunit. However, compounds which are not selective in terms of theirbinding affinity for the α2 and/or α3 subunit relative to the α1 subunitare also encompassed within the scope of the present invention; suchcompounds will desirably exhibit functional selectivity in terms of aselective efficacy for the α2 and/or α3 subunit relative to the α1subunit.

The present invention provides a compound of formula I, or a salt orprodrug thereof: ##STR3## wherein Y represents a methylene (CH₂),carbonyl (C═O) or thiocarbonyl (C═S) linkage;

R¹ and R² independently represent hydrogen, hydrocarbon or aheterocyclic group; or R¹ and R², together with the intervening nitrogenatom, represent an optionally substituted heterocyclic ring selectedfrom azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl,thiomorpholinyl and imidazolyl;

R³ represents hydrogen, hydrocarbon, a heterocyclic group, halogen,cyano, trifluoromethyl, nitro, --OR^(a), --SR^(a), --SOR^(a), --SO₂R^(a), --SO₂ NR^(a) R^(b), --NR^(a) R^(b), --NR^(a) COR^(b), --NR^(a)CO₂ R^(b), --COR^(a), --CO₂ R^(a), --CONR^(a) R^(b) or --CR^(a) ═NOR^(b); and

R^(a) and R^(b) independently represent hydrogen, hydrocarbon or aheterocyclic group.

The present invention also provides a compound of formula I as definedabove, or a salt or prodrug thereof, wherein

R¹ and R² independently represent hydrogen, hydrocarbon or aheterocyclic group; or R¹ and R², together with the intervening nitrogenatom, represent an optionally substituted heterocyclic ring selectedfrom azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl andthiomorpholinyl; and

Y and R³ are as defined above.

Where R¹ and R², together with the intervening nitrogen atom, representan optionally substituted heterocyclic ring, this ring may besubstituted by one or more, preferably one or two, substituents.Examples of optional substituents on the heterocyclic ring include C₁₋₆alkyl and hydroxy. Typical substituents include methyl and hydroxy.

For use in medicine, the salts of the compounds of formula I will bepharmaceutically acceptable salts. Other salts may, however, be usefulin the preparation of the compounds according to the invention or oftheir pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts of the compounds of this invention include acidaddition salts which may, for example, be formed by mixing a solution ofthe compound according to the invention with a solution of apharmaceutically acceptable acid such as hydrochloric acid, sulphuricacid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid,acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid,carbonic acid or phosphoric acid. Furthermore, where the compounds ofthe invention carry an acidic moiety, suitable pharmaceuticallyacceptable salts thereof may include alkali metal salts, e.g. sodium orpotassium salts; alkaline earth metal salts, e.g. calcium or magnesiumsalts; and salts formed with suitable organic ligands, e.g. quaternaryammonium salts.

The term "hydrocarbon" as used herein includes straight-chained,branched and cyclic groups containing up to 18 carbon atoms, suitably upto 15 carbon atoms, and conveniently up to 12 carbon atoms. Suitablehydrocarbon groups include C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇cycloalkyl, C₃₋₇ cycloalkyl(C₁₋₆)alkyl, indanyl, aryl andaryl(C₁₋₆)alkyl.

The expression "a heterocyclic group" as used herein includes cyclicgroups containing up to 18 carbon atoms and at least one heteroatompreferably selected from oxygen, nitrogen and sulphur. The heterocyclicgroup suitably contains up to 15 carbon atoms and conveniently up to 12carbon atoms, and is preferably linked through carbon. Examples ofsuitable heterocyclic groups include C₃₋₇ heterocycloalkyl, C₃₋₇heterocycloalkyl(C₁₋₆)alkyl, heteroaryl and heteroaryl(C₁₋₆)alkylgroups.

Suitable alkyl groups include straight-chained and branched alkyl groupscontaining from 1 to 6 carbon atoms. Typical examples include methyl andethyl groups, and straight-chained or branched propyl, butyl and pentylgroups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl,isobutyl, tert-butyl and 2,2-dimethylpropyl. Derived expressions such as"C₁₋₆ alkoxy", "C₁₋₆ alkylamino" and "C₁₋₆ alkylsulphonyl" are to beconstrued accordingly.

Suitable alkenyl groups include straight-chained and branched alkenylgroups containing from 2 to 6 carbon atoms. Typical examples includevinyl, allyl and dimethylallyl groups.

Suitable alkynyl groups include straight-chained and branched alkynylgroups containing from 2 to 6 carbon atoms. Typical examples includeethynyl and propargyl groups.

Suitable cycloalkyl groups include groups containing from 3 to 7 carbonatoms. Particular cycloalkyl groups are cyclopropyl and cyclohexyl.

Typical examples of C₃₋₇ cycloalkyl(C₁₋₆)alkyl groups includecyclopropylmethyl, cyclohexylmethyl and cyclohexylethyl.

Particular indanyl groups include indan-1-yl and indan-2-yl.

Particular aryl groups include phenyl and naphthyl.

Particular aryl(C₁₋₆)alkyl groups include benzyl, phenylethyl,phenylpropyl and naphthylmethyl.

Suitable heterocycloalkyl groups include azetidinyl, pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl and thiomorpholinyl groups.

Suitable heteroaryl groups include pyridinyl, quinolinyl, isoquinolinyl,pyridazinyl, pyrimidinyl, pyrazinyl, pyranyl, furyl, benzofuryl,dibenzofuryl, thienyl, benzthienyl, pyrrolyl, indolyl, pyrazolyl,indazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl,benzimidazolyl, oxadiazolyl, thiadiazolyl, triazolyl and tetrazolylgroups.

The expression "heteroaryl(C₁₋₆)alkyl" as used herein includesfurylmethyl, furylethyl, thienylmethyl, thienylethyl, oxazolylmethyl,oxazolylethyl, thiazolylmethyl, thiazolylethyl, imidazolylmethyl,imidazolylethyl, oxadiazolylmethyl, oxadiazolylethyl,thiadiazolylmethyl, thiadiazolylethyl, triazolylmethyl, triazolylethyl,tetrazolylmethyl, tetrazolylethyl, pyridinylmethyl, pyridinylethyl,pyrimidinylmethyl, pyrazinylmethyl, quinolinylmethyl andisoquinolinylmethyl.

The hydrocarbon and heterocyclic groups may in turn be optionallysubstituted by one or more groups selected from C₁₋₆ alkyl, adamantyl,phenyl, halogen, C₁₋₆ haloalkyl, C₁₋₆ aminoalkyl, trifluoromethyl,hydroxy, C₁₋₆ alkoxy, aryloxy, keto, C₁₋₃ alkylenedioxy, nitro, cyano,carboxy, C₂₋₆ alkoxycarbonyl, C₂₋₆ alkoxycarbonyl(C₁₋₆)alkyl, C₂₋₆alkylcarbonyloxy, arylcarbonyloxy, aminocarbonyloxy, C₂₋₆ alkylcarbonyl,arylcarbonyl, C₁₋₆ alkylthio, C₁₋₆ alkylsulphinyl, C₁₋₆ alkylsulphonyl,arylsulphonyl, --NR^(v) R^(w), --NR^(v) COR^(w), --NR^(v) CO₂ R^(w),--NR^(v) SO₂ R^(w), --CH₂ NR^(v) SO₂ R^(w), --NHCONR^(v) R^(w),--CONR^(v) R^(w), --SO₂ NR^(v) R^(w) and --CH₂ SO₂ NR^(v) R^(w), inwhich R^(v) and R^(w) independently represent hydrogen, C₁₋₆ alkyl, arylor aryl(C₁₋₆)alkyl.

The term "halogen" as used herein includes fluorine, chlorine, bromineand iodine, especially fluorine.

The present invention includes within its scope prodrugs of thecompounds of formula I above. In general, such prodrugs will befunctional derivatives of the compounds of formula I which are readilyconvertible in vivo into the required compound of formula I.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in Design of Prodrugs,ed. H. Bundgaard, Elsevier, 1985.

Where the compounds according to the invention have at least oneasymmetric centre, they may accordingly exist as enantiomers. Where thecompounds according to the invention possess two or more asymmetriccentres, they may additionally exist as diastereoisomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present invention.

Suitable values for the substituents R¹ and R² include hydrogen, C₁₋₆alkyl, aryl(C₁₋₆)alkyl and heteroaryl(C₁₋₆)alkyl, any of which groupsmay be optionally substituted. Typical substituents include C₁₋₆ alkyl,C₁₋₆ alkoxy and halogen.

Particular values of R¹ and R² include hydrogen, methyl, ethyl andpyridinylmethyl.

Suitably, one of R¹ and R² is other than hydrogen.

Where R¹ and R², together with the intervening nitrogen atom, representan optionally substituted heterocyclic ring, this ring is suitably apiperidinyl, morpholinyl, thiomorpholinyl or imidazolyl ring, any ofwhich rings may be unsubstituted or substituted by one or more,preferably one or two, substituents, typically hydroxy. In this context,typical values for the --NR¹ R² moiety include hydroxy-piperidinyl,morpholinyl, thiomorpholinyl and imidazolyl, preferably morpholinyl.

Suitable values for the substituent R³ include hydrogen, halogen, cyano,nitro, trifluoromethyl, pyrrolyl, furyl, isoxazolyl, amino, C₁₋₆alkylamino, di(C₁₋₆)alkylamino, C₁₋₆ alkyl, C₁₋₆ alkoxy,aryl(C₁₋₆)alkoxy, C₂₋₆ alkylcarbonyl, C₁₋₆ alkylsulphonyl and --CR⁴═NOR⁵, in which R⁴ and R⁵ independently represent hydrogen, methyl orethyl. A particular value of R³ is C₁₋₆ alkyl, especially methyl.

A particular sub-class of compounds according to the invention isrepresented by the compounds of formula II, and salts and prodrugsthereof: ##STR4## wherein Y¹ represents a methylene (CH₂) or carbonyl(C═O) linkage;

R¹³ represents hydrogen, halogen, cyano, nitro, trifluoromethyl,pyrrolyl, furyl, isoxazolyl, amino, C₁₋₆ alkylamino, di(C₁₋₆)akylamino,C₁₋₆ alkyl, C₁₋₆ alkoxy, C₂₋₆ alkylcarbonyl, C₁₋₆ alkylsulphonyl or--CR⁴ ═NOR⁵ ; and

R⁴ and R⁵ independently represent hydrogen, methyl or ethyl.

Suitably, Y¹ represents a carbonyl (C═O) linkage.

Suitably, R¹³ represents C₁₋₆ alkyl, especially methyl.

Specific compounds within the scope of the present invention include:

1-[3-(morpholin-4-ylcarbonyl)phenyl]-5-methylbenzimidazole;

1-[3-(N,N-diethylamido)phenyl]-5-methylbenzimidazole;

1-[3-(4-pyridylmethylamido)phenyl]-5-methylbenzimidazole;

1-[3-(2-pyridylmethylamido)phenyl]-5-methylbenzimidazole;

1-[3-(thiomorpholin-4-ylcarbonyl)phenyl]-5-methylbenzimidazole;

1-[3-(4-hydroxypiperidin-1-ylcarbonyl)phenyl]-5-methylbenzimidazole;

1-[3-(morpholin-4-ylmethyl)phenyl]-5-methylbenzimidazole;

1-[3-(imidazol-1-ylmethyl)phenyl]-5-methylbenzimidazole;

and salts and prodrugs thereof.

Also provided by the present invention is a method for the treatmentand/or prevention of anxiety which comprises administering to a patientin need of such treatment an effective amount of a compound of formula Ias defined above or a pharmaceutically acceptable salt thereof.

Further provided by the present invention is a method for the treatmentand/or prevention of convulsions (e.g. in a patient suffering fromepilepsy or a related disorder) which comprises administering to apatient in need of such treatment an effective amount of a compound offormula I as defined above or a pharmaceutically acceptable saltthereof.

The binding affinity (K_(i)) of the compounds according to the presentinvention for the α3 subunit of the human GABA_(A) receptor isconveniently as measured in the assay described hereinbelow. The α3subunit binding affinity (K_(i)) of the compounds of the invention isideally 10 nM or less, preferably 2 nM or less, and more preferably 1 nMor less.

The compounds according to the present invention will ideally elicit atleast a 40%, preferably at least a 50%, and more preferably at least a60%, potentiation of the GABA EC₂₀ response in stably transfectedrecombinant cell lines expressing the α3 subunit of the human GABA_(A)receptor. Moreover, the compounds of the invention will ideally elicitat most a 30%, preferably at most a 20%, and more preferably at most a10%, potentiation of the GABA EC₂₀ response in stably transfectedrecombinant cell lines expressing the α1 subunit of the human GABA_(A)receptor.

The potentiation of the GABA EC₂₀ response in stably transfected celllines expressing the α3 and α1 subunits of the human GABA_(A) receptorcan conveniently be measured by procedures analogous to the protocoldescribed in Wafford et al., Mol. Pharmacol., 1996, 50, 670-678. Theprocedure will suitably be carried out utilising cultures of stablytransfected eukaryotic cells, typically of stably transfected mouse Ltk⁻fibroblast cells.

The compounds according to the present invention exhibit anxiolyticactivity, as demonstrated by a positive response in the elevated plusmaze and conditioned suppression of drinking tests (cf. Dawson et al.,Psychopharmacology, 1995, 121, 109-117). Moreover, the compounds of theinvention are substantially non-sedating, as confirmed by an appropriateresult obtained from the response sensitivity (chain-pulling) test (cf.Bayley et al., J. Psychopharmacol., 1996, 10, 206-213).

The compounds according to the present invention may also exhibitanticonvulsant activity. This can be demonstrated by the ability toblock pentylenetetrazole-induced seizures in rats and mice, following aprotocol analogous to that described by Bristow et al. in J. Pharmacol.Exp. Ther., 1996, 279, 492-501.

In order to elicit their behavioural effects, the compounds of theinvention will ideally be brain-penetrant; in other words, thesecompounds will be capable of crossing the so-called "blood-brainbarrier". Preferably, the compounds of the invention will be capable ofexerting their beneficial therapeutic action following administration bythe oral route.

The invention also provides pharmaceutical compositions comprising oneor more compounds of this invention in association with apharmaceutically acceptable carrier. Preferably these compositions arein unit dosage forms such as tablets, pills, capsules, powders,granules, sterile parenteral solutions or suspensions, metered aerosolor liquid sprays, drops, ampoules, auto-injector devices orsuppositories; for oral, parenteral, intranasal, sublingual or rectaladministration, or for administration by inhalation or insufflation. Forpreparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a pharmaceutically acceptable saltthereof. When referring to these preformulation compositions ashomogeneous, it is meant that the active ingredient is dispersed evenlythroughout the composition so that the composition may be readilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules. This solid preformulation composition is thensubdivided into unit dosage forms of the type described above containingfrom 0.1 to about 500 mg of the active ingredient of the presentinvention. Typical unit dosage forms contain from 1 to 100 mg, forexample 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. Thetablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavoured syrups, aqueous or oilsuspensions, and flavoured emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatin.

In the treatment of anxiety, a suitable dosage level is about 0.01 to250 mg/kg per day, preferably about 0.05 to 100 mg/kg per day, andespecially about 0.05 to 5 mg/kg per day. The compounds may beadministered on a regimen of 1 to 4 times per day.

The compounds in accordance with the present invention may be preparedby a process which comprises reacting a compound of formula III:##STR5## wherein Y, R¹, R² and R³ are as defined above; with formicacid, typically at an elevated temperature.

In another procedure, the compounds according to the present inventionin which Y represents a methylene linkage may be prepared by a processwhich comprises treating a compound of formula IV: ##STR6## wherein R¹,R² and R³ are as defined above; with a reducing agent such as lithiumaluminium hydride.

In a further procedure, the compounds according to the present inventionin which Y represents a thiocarbonyl linkage may be prepared by aprocess which comprises treating the corresponding compound of formulaIV as defined above with Lawesson's reagent[2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulphide]or phosphorus pentasulphide in a suitable solvent, e.g. pyridine, atambient or elevated temperatures, suitably at the reflux temperature ofthe solvent.

In a yet further procedure, the compounds according to the presentinvention in which Y represents a methylene linkage may be prepared by aprocess which comprises reacting a compound of formula V with a compoundof formula VI: ##STR7## wherein R¹, R² and R³ are as defined above, andL represents a suitable leaving group.

The leaving group L is suitably a halogen atom, e.g. chloro, in whichcase the reaction between compounds V and VI is conveniently carried outby stirring the reactants in a suitable solvent, for exampleN,N-dimethylformamide, tetrahydrofuran, acetonitrile or dichloromethane,optionally in the presence of a base such as potassium carbonate ortriethylamine.

In a still further procedure, the compounds according to the inventionin which Y represents a carbonyl linkage, i.e. the compounds of formulaIV as defined above, may be prepared by a process which comprisesreacting a compound of formula VI as defined above with a compound offormula VII: ##STR8## wherein R³ is as defined above, and Q represents areactive carboxylate moiety.

Suitable values for the reactive carboxylate moiety Q include esters,for example C₁₋₄ alkyl esters; acid anhydrides, for example mixedanhydrides with C₁₋₄ alkanoic acids; acid halides, for example acidchlorides; and acylimidazoles.

By way of example, the intermediates of formula VII above wherein Q isan acid chloride moiety may be prepared by treating the correspondingcarboxylic acid derivative with thionyl chloride in toluene. Similarly,the intermediates of formula VII wherein Q is an acylimidazole moietymay be prepared by treating the corresponding carboxylic acid derivativewith 1,1'-carbonyldiilmidazole. Alternatively, the reactive carboxylatemoiety Q may be obtained by treating the corresponding compound whereinQ is carboxy with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride and 1-hydroxybenzotriazole hydrate, optionally in thepresence of triethylamine; the resulting activated carboxylateintermediate may then suitably be reacted in situ with the requiredcompound of formula VI.

Typical intermediates of formula V above wherein the leaving group L isa halogen atom may suitably be prepared by treating the appropriatecompound of formula VII wherein Q represents carboxy with a reducingagent, for example lithium aluminium hydride, followed by conversion ofthe primary hydroxy group in the compound thereby obtained into ahalogen atom by treatment with a thionyl halide such as thionylchloride.

The intermediates of formula VII above may suitably be prepared byreacting a compound of formula VIII: ##STR9## wherein R³ and Q are asdefined above; with formic acid, typically at an elevated temperature.

The intermediates of formula III and VIII above may be prepared bymethods analogous to those described in EP-A-0616807.

Where they are not commercially available, the starting materials offormula VI may be prepared by methods analogous to those described inthe accompanying Examples, or by standard methods well known from theart.

It will be understood that any compound of formula I initially obtainedfrom any of the above processes may, where appropriate, subsequently beelaborated into a further compound of formula I by techniques known fromthe art. Indeed, as noted above, the intermediates of formula IV arecompounds according to the invention in their own right.

Where the above-described processes for the preparation of the compoundsaccording to the invention give rise to mixtures of stereoisomers, theseisomers may be separated by conventional techniques such as preparativechromatography. The novel compounds may be prepared in racemic form, orindividual enantiomers may be prepared either by enantiospecificsynthesis or by resolution. The novel compounds may, for example, beresolved into their component enantiomers by standard techniques such aspreparative HPLC, or the formation of diastereomeric pairs by saltformation with an optically active acid, such as(-)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-1-tartaricacid, followed by fractional crystallization and regeneration of thefree base. The novel compounds may also be resolved by formation ofdiastereomeric esters or amides, followed by chromatographic separationand removal of the chiral auxiliary.

During any of the above synthetic sequences it may be necessary and/ordesirable to protect sensitive or reactive groups on any of themolecules concerned. This may be achieved by means of conventionalprotecting groups, such as those described in Protective Groups inOrganic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W.Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, JohnWiley & Sons, 1991. The protecting groups may be removed at a convenientsubsequent stage using methods known from the art.

The following Examples illustrate the preparation of compounds accordingto the invention.

The compounds in accordance with this invention potently inhibit thebinding of [³ H]-flumazenil to the benzodiazepine binding site of humanGABA_(A) receptors containing the α2 or α3 subunit stably expressed inLtk⁻ cells.

Reagents

Phosphate buffered saline (PBS).

Assay buffer: 10 mM KH₂ PO₄, 100 mM KCl, pH 7.4 at room temperature.

[³ H]-Flumazenil (18 nM for α1β3γ2 cells; 18 nM for α2β3γ2 cells; 10 nMfor α3β3γ2 cells) in assay buffer.

Flunitrazepam 100 μM in assay buffer.

Cells resuspended in assay buffer (1 tray to 10 ml).

Harvesting Cells

Supernatant is removed from cells. PBS (approximately 20 ml) is added.The cells are scraped and placed in a 50 ml centrifuge tube. Theprocedure is repeated with a further 10 ml of PBS to ensure that most ofthe cells are removed. The cells are pelleted by centrifuging for 20 minat 3000 rpm in a benchtop centrifuge, and then frozen if desired. Thepellets are resuspended in 10 ml of buffer per tray (25 cm×25 cm) ofcells.

Assay

Can be carried out in deep 96-well plates or in tubes. Each tubecontains:

300 μl of assay buffer.

50 μl of [³ H]-flumazenil (final concentration for α1β3γ2: 1.8 nM; forα2β3γ2: 1.8 nM; for α3β3γ2: 1.0 nM).

50 μl of buffer or solvent carrier (e.g. 10% DMSO) if compounds aredissolved in 10% DMSO (total); test compound or flunitrazepam (todetermine non-specific binding), 10 μM final concentration.

100 μl of cells.

Assays are incubated for 1 hour at 40° C., then filtered using either aTomtec or Brandel cell harvester onto GF/B filters followed by 3×3 mlwashes with ice cold assay buffer. Filters are dried and counted byliquid scintillation counting. Expected values for total binding are3000-4000 dpm for total counts and less than 200 dpm for non-specificbinding if using liquid scintillation counting, or 1500-2000 dpm fortotal counts and less than 200 dpm for non-specific binding if countingwith meltilex solid scintillant. Binding parameters are determined bynon-linear least squares regression analysis, from which the inhibitionconstant K_(i) can be calculated for each test compound.

The compounds of the accompanying Examples were tested in the aboveassay, and all were found to possess a K_(i) value for displacement of[³ H]-flumazenil from the α2 and/or α3 subunit of the human GABA_(A)receptor of 100 nM or less.

EXAMPLE 1 1-[3-(Morpholin-4-ylcarbonyl)phenyl]-5-methylbenzimidazole

Step 1: 4-Methyl-3'-carboxy-2-nitrodiphenylamine

4-Methyl-2-nitroaniline, 3-iodobenzoic acid (10 g, 40 mmol), potassiumcarbonate (5.5 g, 40 mmol) and a catalytic amount of CuI were thoroughlymixed and heated to 230° C. for 4 hours. The reaction mixture wasallowed to cool to 100° C. and water added. After cooling to roomtemperature the solution was rendered acidic by careful addition ofglacial acetic acid. The precipitate was filtered off and washed withdichloromethane. Recrystallization from 2-propanol afforded product.Yield 4.3 g.

Step 2: 2-Amino-3'-carboxy-4-methyldiphenylamine

A mixture of 4-methyl-3'-carboxy-2-nitrodiphenylamine (1 g, 3.11 mmol)and palladium on activated carbon (5%, 0.1 g) in MeOH (25 ml) washydrogenated at ambient pressure until the hydrogen uptake had ceased.The reaction mixture was filtered through celite into a few millilitresof ethereal hydrogen chloride. Evaporation of solvent left the desiredproduct (0.95 g, 2.89 mmol).

Step 3: 1-(3-Carboxyphenyl)-5-methylbenzimidazole

A mixture of 2-amino-3'-carboxy-4-methyldiphenylamine (6.00 g, 14.4mmol) and formic acid (60 ml) was refluxed for 16 h. After evaporationto dryness, the residue was dissolved in ethyl acetate (100 ml) andwashed with water (100 ml). The organic phase was dried and evaporated.The crude product was purified by column chromatography with methylenechloride as the eluent. Yield 4.2 g. ¹ H NMR (250 MHz, DMSO) δ 2.47 (3H,s), 7.21 (1H, dd, J=1.4 Hz), 7.53 (1H, d, J=8.3 Hz), 7.60 (1H, s), 7.75(1H, t, J=7.8 Hz), 7.95 (1H, m), 8.03 (1H, m), 8.14 (1H, m) and 8.62(1H, s). MS M⁺ 253.

Step 4: 1-[3-(Morpholin-4-ylcarbonyl)phenyl]-5-methylbenzimidazole

A mixture of 1-(3-carboxyphenyl)-5-methylbenzimidazole (50 mg, 0.19 mM),1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, HCl (76 mg, 0.39 mM),hydroxybenzotriazole (53 mg, 0.39 mM) in dimethylformamide (2 ml) wastreated with triethylamine (80 μl, 0.39 mM) and morpholine (20 μl, 0.22mM) and the reaction stirred for 18 hours at room temperature under N₂.Dilution with water and extractive work up with ethyl acetate wasfollowed by chromatography on silica gel. Yield 37 mg. ¹ H NMR (360 MHz,DMSO) 2.51 (3H, s), 3.47-3.73 (8H, m), 7.16 (1H, d, J=8.3 Hz), 7.41 (1H,d, J=8.3 Hz), 7.45 and 7.47 (1H, dt, J=1.4 Hz, 7.0 Hz), 7.57-7.60 (3H,m), 7.66 (1H, s) and 8.08 (1H, s). MS M⁺ 322.

EXAMPLE 2 1-[3-(N,N-Diethylamido)phenyl]-5-methylbenzimidazole

Prepared by an analogous procedure to that described in Example 1 usingdiethylamine. ¹ H NMR (360 MHz, CDCl₃) 1.10-1.24 (6H, m), 2.51 (3H, s),3.27-3.42 (2H, m), 3.50-3.64 (2H, m), 7.16 (1H, d, J=8.3 Hz), 7.42-7.45(2H, m), 7.53-7.62 (3H, m), 7.66 (1H, s) and 8.08 (1H, s). MS M⁺ 308.

EXAMPLE 3 1-[3-(4-Pyridylmethylamido)phenyl]-5-methylbenzimidazole

Prepared by an analogous procedure to that described in Example 1 using4-pyridylmethylamine. ¹ H NMR (360 MHz, CDCl₃) 2.44 (3H, s), 4.68 (2H,d, J=5.9 Hz), 7.09 (1H, d, J=8.3 Hz), 7.25 (1H, d, J=6.0 Hz), 7.35 (1H,d, J=8.3 Hz), 7.49 (1H, s), 7.61-7.66 (2H, m), 7.88 (1H, s), 7.97-8.01(2H, m), 8.16 (1H, br t) and 8.49 (2H, m). MS M⁺ 343.

EXAMPLE 4 1-[3-(2-Pyridylmethylamido)phenyl]-5-methylbenzimidazole

Prepared by an analogous procedure to that described in Example 1 using2-pyridylmethylamine. ¹ H NMR (250 MHz, DMSO) 2.45 (3H, s), 4.61 (2H, d,J=5.9 Hz), 7.19 (1H, d, J=8.7 Hz), 7.26 (1H, dd, J=4.8 and 12.3 Hz),7.36 (1H, d, J=7.8 Hz), 7.55-7.59 (2H, m), 7.71-7.80 (2H, m), 7.88 (1H,d, J=8.9 Hz), 7.99-8.02 (1H, d, J=7.7 Hz), 8.18 (1H, s), 8.30-8.35 (1H,m), 8.58 (1H, s) and 9.30 (1H, br t, J=5.9 Hz). MS M⁺ 343.

EXAMPLE 5 1-[3-(Thiomorpholin-4-ylcarbonyl)phenyl]-5-methylbenzimidazole

Prepared by an analogous procedure to that described in Example 1 usingthiomorpholine. ¹ H NMR (250 MHz, DMSO) 2.45 (3H, s), 2.67 (4H, br s),3.63 (2H, br), 3.88 (2H, br), 7.18 (1H, d, J=8.3 Hz), 7.46-7.57 (3H, m),7.66-7.78 (3H, m) and 8.55 (1H, s). MS M⁺ 338.

EXAMPLE 61-[3-(4-Hydroxypiperidin-1-ylcarbonyl)phenyl]-5-methylbenzimidazole

Prepared by an analogous procedure to that described in Example 1 using4-hydroxypiperidine. ¹ H NMR (250 MHz, DMSO) 1.39 (2H, br), 1.77 (2H,br), 2.45 (3H, s), 3.24 (1H, br), 3.52 (2H, br), 3.72 (2H, br), 4.82(1H, d, J=3.9 Hz), 7.17 (1H, d, J=8.3 Hz), 7.45-7.57 (3H, m), 7.65-7.74(3H, m) and 8.54 (1H, s). MS M⁺ 336.

EXAMPLE 7 1-[3-(Morpholin-4-ylmethyl)phenyl]-5-methylbenzimidazole

Step 1: 1-(3-Hydroxymethylphenyl)-5-methylbenzimidazole

1-(3-Carboxyphenyl)-5-methylbenzimidazole (2 g, 0.079 mM) was dissolvedin dry tetrahydrofuran (100 ml) and cooled to 0° C. Lithium aluminiumhydride (7.9 ml, 1.0 M solution) was added dropwise over 10 minutes.After complete addition the reaction was heated to reflux for 4 hours.The reaction was cooled and quenched by addition of water (2 ml) and 2Nsodium hydroxide (4 ml). The reaction was diluted with ethyl acetate andfiltered through hyflo, washing with ethyl acetate. Dried over magnesiumsulphate and evaporated (1.2 g). MS M⁺ 239.

Step 2: 1-(3-Chloromethylphenyl)-5-methylbenzimidazole HCl

1-(3-Hydroxymethylphenyl)-5-methylbenzimidazole (0.3 g, 1.26 mM) wasdissolved in dichloromethane (3 ml). Thionyl chloride (92 μl, 1.26 mM)was added and the reaction stirred for 30 minutes. Solvent evaporated toobtain product (0.36 g). MS M⁺ 957.

Step 3: 1-[3-(Morpholin-4-ylmethyl)phenyl]-5-methylbenzimidazole

1-(3-Chloromethyl)phenyl-5-methylbenzimidazole (0.11 g, 0.37 mM) wasdissolved in dimethylformamide (5 ml). Potassium carbonate (52 mg, 0.37mM) was added followed by morpholine (97 μl, 1.11 mM) and the reactionheated to 80° C. for 18 hours. Dilution with water and extraction withethyl acetate was followed by chromatography over silica gel (61 mg). ¹H NMR (360 MHz, DMSO, TFA) 2.54 (3H, s), 3.17 (2H, br), 3.31 (2H, br),3.18 (2H, br), 3.95 (2H, br), 4.50 (2H, s), 7.49 (1H, d, J=5.7 Hz).7.78-7.94 (5H, m), 8.08 (1H, s) and 9.94 (1H, s). MS M⁺ 308.

EXAMPLE 8 1-[3-(Imidazol-1-ylmethyl)phenyl]5-methylbenzimidazole

1-(3-Chloromethylphenyl)-5-methylbenzimidazole (0.1 g, 0.34 mM) wasdissolved in dichloromethane (5 ml), imidazole (92 mg, 1.36 mM) wasadded and the reaction heated to reflux for 18 hours, then diluted withwater and extracted into ethyl acetate, dried (MgSO₄) and purified bysilica chromatography. Yield 34 mg. ¹ H NMR (250 Mz, DMSO) δ 2.44 (3H,s), 5.32 (2H, s), 6.92 (1H, t, J=0.9 Hz), 7.16 (1H, d, J=8.3 Hz),7.29-7.35 (2H, m), 7.47 (1H, d, J=8.3 Hz), 7.57-7.61 (4H, m), 7.83 (1H,t, J=0.9 Hz) and 8.49 (1H, s). MS m/e 289.

What is claimed is:
 1. A compound represented by formula II, and saltsthereof: ##STR10## wherein Y¹ represents a methylene (CH₂) or carbonyl(C═O) linkage;R¹³ represents a hydrogen, halogen, cyano, nitro,trifluoromethyl, pyrrolyl, furyl, isoxazolyl, amino, C₁₋₆ alkylamino,C₁₋₆ alkyl, C₁₋₆ alkoxy, C₂₋₆ alkylcarbonyl, C₁₋₆ alkylsulphonyl or--CR⁴ ═NOR⁵ ; and R⁴ and R⁵ independently represent hydrogen, methyl orethyl.
 2. A compound as claimed in claim 1 wherein Y¹ represents acarbonyl (C═O) linkage.
 3. A compound as claimed in claim 1 wherein R¹³represents C₁₋₆ alkyl.
 4. A compound selectedfrom:1-[3-(morpholin-4-ylcarbonyl)phenyl]-5-methylbenzimidazole;1-[3-(morpholin-4-ylmethyl)phenyl]-5-methylbenzimidazole; and saltsthereof.
 5. A pharmaceutical composition comprising a compound offormula II as defined in claim 1 or a pharmaceutically acceptable saltthereof in association with a pharmaceutically acceptable carrier.
 6. Amethod for the treatment of anxiety which comprises administering to apatient in need of such treatment an effective amount of a compound offormula II as defined in claim 1 or a pharmaceutically acceptable saltthereof.
 7. A method for the treatment of convulsions which comprisesadministering to a patient in need of such treatment an effective amountof a compound of formula II as defined in claim 1 or a pharmaceuticallyacceptable salt thereof.